一种基于微流控芯片技术的流式细胞计数系统

采用聚二甲基硅氧烷（PDMS）材料制作微流控流式细胞计数芯片,利用负压驱动与鞘液夹流技术实现样品的水力聚焦,达到了10μm的样品聚焦宽度。基于激光诱导荧光技术,制作了小型化的检测装置。以488nm固体激光器为光源,激光束以45°方向穿过一个水平狭缝,以线光源形式汇聚到微流控芯片的检测区域,并与微通道垂直交叉,细胞样品的荧光信号通过光电倍增管收集。整个分析系统结构简单,操作方便,灵敏度高,可初步实现细胞的计数。     

An integrated microfluidic signal generator using multiphase droplet grating

An integrated and reconfigurable optofluidic signal generator based on multiphase droplet grating is demonstrated in this paper. The chip is fabricated with an inexpensive, optically clear and non-toxic silicone elastomer-polydimethylsiloxane (PDMS) by conventional soft lithography. Droplet grating is formed by a stream of plugs which are generated through a typical microfluidic T-junction. Since the refractive indices of the two immiscible liquids are different, the alternative mobility of the plug results in the periodical change of the reflectivity at the fluid/PDMS interface. The real-time tunability in the frequency and amplitude of the signal can be realized by varying the flow rates of the liquids. In experiments, both rectangle and triangle signals are displayed and the signal frequency ranges from 1 to 525 Hz. This signal generator can be easily integrated into other microfluidic networks to create versatile functionalities. Furthermore, we present coding functions based on the signal generator on a chip. Such a signal generator has great potential as a signal source or a part of functionalities for lab-on-a-chip applications.     

An improved bulk acoustic waves chip based on a PDMS bonding layer for high-efficient particle enrichment

In this work, we developed a feasible way to package bulk acoustic waves chip with sandwich structure by inserting a polydimethylsiloxane (PDMS) layer as the adhesive between cover glass and silicon substrate. After spin-coating and curing process, a PDMS layer was formed on one side of the cover glass and then bonded to the silicon substrate with microchannels by oxygen plasma treating. Both simulation and experiment showed that the chip was not leaking and the acoustic waves produced by the piezoelectric transducer could be propagated through the PDMS layer. Finally, a standing wave field was formed in the microchannels. Compared with traditional chip bonded by anodic bonding, simulation results showed that this packaging method did decrease the acoustic pressure in the channel, but the reduction was acceptable. After optimizing the experimental parameters, we successfully aggregated 15-μm silica spheres under a very low input power (21 dBm) at a flow velocity of 1 ml/h, and the enrichment efficiency of silica spheres was greater than 97%.     

单泵控制聚焦流形态的微流控芯片的研制

研究一种单动力源、聚焦流形态可控的用于细胞排队的微流控芯片。建立了样品沟道与鞘流沟道不同长度比例、不同夹角的模型并进行了不同负压条件下聚焦流形态仿真,运用SPSS软件进行了回归分析并进行了模型优化。在芯片的微加工过程中,利用印刷电路板(PCB)制作了母板,以聚二甲基硅氧烷(PDMS)为芯片主要材料,制作了PDMS—PDMS,PDMS—玻璃及PCB—PDMS三种芯片。制作的芯片能够在单个动力源条件下控制聚焦流宽度,使不同大小的微粒及细胞呈单个排列流动。研究结果为分析不同尺寸的细胞而选择合适的样品流沟道与鞘流沟道长度、夹角等条件提供了依据,所制作的芯片也达到了廉价且实用的目的。     

Determination of dynamic contact angles within microfluidic devices

Here, we report a single-point detection method for the determination of dynamic surface conditions inside microfluidic channels. The proposed method is based on monitoring fluorescence amplitude as a function of the convolution of a laser beam with segmented flow consisting of two immiscible liquids, one containing fluorescent dye. The fluorescence amplitude is determined by the flow rate and the droplet shape, which is affected by the channel surface properties. We modeled the interaction of a droplet and a laser beam via computer-aided design software, using the laser beam location in relation to the droplet shape as a parameter. The method was applied to fused silica capillaries with both unmodified and modified surfaces, with segmented flow exhibiting water contact angles of ≈?30° and ≈?100°, respectively. The method allows discrimination between hydrophillic and hydrophobic surfaces, as well as the quality of the treatment. The results were verified using fluorescence imaging of the droplets via a stroboscopic technique. We also applied this method to the analysis of microfabricated channels with non-circular cross sections. We demonstrated that the technique enables the determination of the hydrophobicity of channel surfaces, a crucial property required for the generation of segmented flow or emulsions for applications such as digital PCR.     

Viscosity-difference-induced asymmetric selective focusing for large stroke particle separation

We developed a new approach for particle separation by introducing viscosity difference of the sheath flows to form an asymmetric focusing of sample particle flow. This approach relies on the high-velocity gradient in the asymmetric focusing of the particle flow to generate a lift force, which plays a dominated role in the particle separation. The larger particles migrate away from the original streamline to the side of the higher relative velocity, while the smaller particles remain close to the streamline. Under high-viscosity (glycerol–water solution) and low-viscosity (PBS) sheath flows, a significant large stroke separation between the smaller (1.0 μm) and larger (9.9 μm) particles was achieved in a sample microfluidic device. We demonstrate that the flow rate and the viscosity difference of the sheath flows have an impact on the interval distance of the particle separation that affects the collected purity and on the focusing distribution of the smaller particles that affects the collected concentration. The interval distance of 293 μm (relative to the channel width: 0.281) and the focusing distribution of 112 μm (relative to the channel width: 0.107) were obtained in the 1042-μm-width separation area of the device. This separation method proposed in our work can potentially be applied to biological and medical applications due to the wide interval distance and the narrow focusing distribution of the particle separation, by easy manufacturing in a simple device.     

An integrated flow-cell for full sample stream control

In this study, we present a novel three-dimensional hydrodynamic sheath flow chip that allows full control of a sample stream. The chip offers the possibility to steer each of the four side sheath flows individually. The design of the flow-cell exhibits high flexibility in creating different sample stream profiles (width and height) and allows navigation of the sample stream to every desired position inside the microchannel (vertical and horizontal). This can be used to bring the sample stream to a sensing area for analysis, or to an area of actuation (e.g. for cell sorting). In addition, we studied the creation of very small sample stream diameters. In microchannels (typically 25 × 40 μm2), we created sample stream diameters that were five to ten times smaller than the channel dimensions, and the smallest measured sample stream width was 1.5 μm. Typical flow rates are 0.5 μl/min for the sample flow and around 100 μl/min for the cumulated sheath flows. The planar microfabricated chip, consisting of a silicon–glass sandwich with an intermediate SU-8 layer, is much smaller (6 × 9 mm2) than the previously presented sheath flow devices, which makes it also cost-effective. We present the chip design, fluidic simulation results and experiments, where the size, shape and position of the sample stream have been established by laser scanning confocal microscopy and dye intensity analysis.     

Micro molecular tagging velocimetry for analysis of gas flows in mini and micro systems

A new micro molecular tagging velocimetry (μMTV) setup has been developed to analyze velocity fields in confined internal gas flows. MTV is a little-intrusive velocimetry technique. It relies on the properties of molecular tracers which can experience relatively long lifetime luminescence once excited by a laser beam with an appropriate wavelength. The technique has been validated for acetone seeded flows of argon inside a 1 mm depth rectangular minichannel, with a multilayer design offering two optical accesses. Velocity profiles have been obtained using a specific data reduction process, with a resolution in the order of 15 μm. The experimental data are compared to theoretical velocity profiles of compressible pressure-driven flows. A good agreement is observed, except close to the walls, where the accuracy would still need to be improved. Following these first results obtained at atmospheric pressure, the influence of pressure on the luminescence intensity of acetone molecules is analyzed. The obtained data lead to a discussion of MTV applicability to rarefied flows and its possible use for a direct measurement of velocity slip at the channel walls.     

Use of a porous membrane for gas bubble removal in microfluidic channels: physical mechanisms and design criteria

We demonstrate and explain a simple and efficient way to remove gas bubbles from liquid-filled microchannels, by integrating a hydrophobic porous membrane on top of the microchannel. A prototype chip is manufactured in hard, transparent polymer with the ability to completely filter gas plugs out of a segmented flow at rates up to 7.4 μl/s/mm² of membrane area. The device involves a bubble generation section and a gas removal section. In the bubble generation section, a T-junction is used to generate a train of gas plugs into a water stream. These gas plugs are then transported toward the gas removal section, where they slide along a hydrophobic membrane until complete removal. The system has been successfully modeled, and four necessary operating criteria have been determined to achieve a complete separation of the gas from the liquid. The first criterion is that the bubble length needs to be larger than the channel diameter. The second criterion is that the gas plug should stay on the membrane for a time sufficient to transport all the gas through the membrane. The third criterion is that the gas plug travel speed should be lower than a critical value: otherwise a stable liquid film between the bubble and the membrane prevents mass transfer. The fourth criterion is that the pressure difference across the membrane should not be larger than the Laplace pressure to prevent water from leaking through the membrane.     

Illumination and scattered light detection using a light source consisting of sub-micrometer defect arrays formed on a extremely flat light waveguide core

We have previously argued that an optical sensor combined total analysis system (TAS) is one of the indispensable functional components needed to realize a “ubiquitous human healthcare” system. To achieve this goal, we have proposed a fundamental structure for illuminating a minute cell or particle running along a microfluidic channel using a flat waveguide construction. It is desirable that the TAS light source should be arranged as close to the specimen flow as possible in order to acquire the necessary optical properties; hence, artificial defects formed on the surface of a flat light waveguide are considered to be a promising candidate for realizing the arbitrary-shaped light source for a highly functional optical TAS structure. Based on this idea, we fabricated a structure, constructing a flat and square light source consisting of rectangular solids, sub-micrometer in size, with a 1-μm thick and a 12-μm wide light waveguide core. We successfully trial-manufactured an optical TAS chip with a fluidic channel containing a 14 × 10-μm cross section, and an extremely flat light waveguide core. We repeatedly confirmed that the defect array could function as an approximately square light source when a 650-nm wavelength laser power was carefully introduced. Furthermore, we developed a hybrid numerical calculation method base on the finite-difference, time-domain method together with the beam propagation method. Utilizing this hybrid method, we evaluated the optical response when a particle runs across the light source while changing the aperture length of a shading mask to obtain signals with both higher intensity and shorter full width at half maximum. The numerical results were compared with experimental results obtained using an image acquisition system, and demonstrated good qualitative accord.     

A microfluidic chip of multiple-channel array with various oxygen tensions for drug screening

Oxygen participates in numbers of cellular activities and behaviors in both normal and pathological tissues. In physiological microenvironment, oxygen tension is generally below 21 % and varies in different species, states and regions of organs. However, present studies of cellular behavior in vitro are performed in an ambient level, which is not conformity to the reality in vivo. In this study, a microfluidic device was developed to generate controllable oxygen tensions on a multiple-channel array chip for high-throughput drug screening. Controlling various concentrations of chemical reagents with confined flow rate, specific oxygen tensions can be established from 1.6 to 21 %, where the oxygen tension of each channel can be modulated in demand. When the concentrations of pyrogallol change from 100 to 700 μg/mL with the flow rate of 5 μL/min, oxygen tensions in cell chambers range from 12.5 to 3.87 %. Pyrogallol with the concentration of 0 μg/mL is used as the control group to obtain 20.9 % oxygen condition. The developed microfluidic chip was used to investigate the cytotoxicity of TPZ and cisplatin, and the results demonstrate different manners of two oxygen-sensitive anti-tumor drugs in oxygen-dependent cytotoxic responses. Due to its character, the microfluidic device is believed to establish any desired and measurable oxygen tension distribution for pharmacology development, which is promising to improve efficiency and reduce tedious operation for pharmaceutical studies.     

Magnetically controlled valve for flow manipulation in polymer microfluidic devices

A simple, external in-line valve for use in microfluidic devices constructed of polydimethylsiloxane (PDMS) is described. The actuation of the valve is based on the principle that flexible polymer walls of a liquid channel can be pressed together by the aid of a permanent magnet and a small metal bar. In the presence of a small NdFeB magnet lying below the channel of interest, the metal bar is pulled downward simultaneously pushing the thin layer of PDMS down thereby closing the channel stopping any flow of fluid. The operation of the valve is dependent on the thickness of the PDMS layer, the height of the channel, the gap between the chip and the magnet and the strength of the magnet. The microfluidic channels are completely closed to fluid flows ranging from 0.1 to 1.0 μL/min commonly used in microfluidic applications.     

A positive pressure-driven PDMS pump for fluid handling in microfluidic chips

By utilizing the high gas permeability of polydimethylsiloxane (PDMS), a simple positive pressure-driven pumping method was introduced. The pump was an aerated PDMS with a central channel in it and packing with a transfusion bottle. It could be attached to the inlet of microfluidic chip using a Teflon tube to release the air into the microfluidic system and then to create a positive pressure for driving fluid. In comparison with the degas-based PDMS pump, positive pressure-driven PDMS pump offered increased system flexibility and reduced individual device fabrication complexity due to its independence and versatility. More importantly, it offered the advantages that the PDMS pump could be wrapped in transfusion bottles to meet the readily available requirements, and it also easily assembled, which only required the user use a Teflon tube to connect a PDMS pump and a microfluidic chip. This assembly provided great freedom to meet different pumping requirements. Furthermore, this PDMS pump could offer many possible configures of pumping power by adjusting the geometries of the pump or by combining different pump modules, the adjustment of pumping capacity was investigated. To help design pumps with a suitable pumping performance, the sealing effect, pumping pressure and flow rate were also investigated. The results indicated that the performance of the positive pressure-driven PDMS pump was reliable. Finally, we demonstrated the utility of this pumping method by applying it to a PDMS-based viscometer microfluidic chip.     

Microfluidic fluorescence-activated cell sorting (μFACS) chip with integrated piezoelectric actuators for low-cost mammalian cell enrichment

A low-cost, microfluidic fluorescence-activated cell sorting (μFACS) microchip integrated with two piezoelectric lead–zirconate–titanate actuators was demonstrated for automated, high-performance mammalian cell analysis and enrichment. In this PDMS–glass device, cells were hydrodynamically focused into a single file line in the lateral direction by two sheath flows, and then interrogated with a forward scattering and confocal fluorescent detection system. The selected cells were displaced transversely into a collection channel by two piezoelectric actuators that worked in a pull–push relay manner with a minimal switching time of ~0.8 ms. High detection throughput (~2500 cells/s), high sorting rate (~1250 cells/s), and high sorting efficiency (~98%) were successfully achieved on the μFACS system. Six cell mixture samples containing 22.87% of GFP-expressing HeLa cells were consecutively analyzed and sorted on the chip, revealing a stable sorting efficiency of 97.7 ± 0.93%. In addition, cell mixtures containing 37.65 and 3.36% GFP HeLa cells were effectively enriched up to 83.82 and 78.51%, respectively, on the microchip, and an enrichment factor of 105 for the low-purity (3.36%) sample was successfully obtained. This fully enclosed, disposable microfluidic chip provides an automated platform for low-cost fluorescence-based cell detection and enrichment, and is attractive to applications where cross-contamination between runs and aerosol hazard are the primary concerns.     

Investigation on pressure drop characteristic and mass transfer performance of gas–liquid flow in micro-channels

Pressure drop characteristics and mass transfer performance of gas–liquid two-phase flow in micro-channels with different surface wettabilities were experimentally investigated. Side-entry T micro-channel mixers made of glass and polydimethylsiloxane were tested. Frictional pressure drop was found to decrease as the hydrophobicity of the channel surface increased. The flow patterns observed in the experiment were classified as slug flow and continuous gas phase flows. The modified Hagen–Poiseuille equation and Lockhart–Martinelli model were developed to predict the pressure drop for these two types of flow, respectively. The effect of surface wettability was heuristically incorporated in the present models which can correlate well the experimental results. Mass transfer performance was studied by the physical absorption of oxygen into de-ionized water. The results show that the volumetric mass transfer coefficients in hydrophobic micro-channels are generally higher than those in hydrophilic ones. The empirical correlations of overall volumetric mass transfer coefficients were proposed.     

A microfluidic optical beam steerer

This paper reports the fabrication and characterization of a prototype microfluidic device that can act as a periodic beam steerer. The prototype is formed by a simple T-junction followed by a serpentine channel that allows generation of a periodical segmented flow of air and water bubbles. If light hits the channel wall with a suitable angle, it can be either transmitted or reflected by the segmented flow, giving rise to an alternating beam steerer. The duty cycle, switching frequency, and overall stability and reproducibility of this prototype system are presented and discussed.     

A silicone-based microfluidic chip grafted with carboxyl functionalized hyperbranched polyglycerols for selective protein capture

A poly(dimethylsiloxane) (PDMS)-based functional microfluidic device containing a charged matrix of PDMS pillar arrays grafted with hyperbranched polyglycerols (HPGs) was developed. Samples of PDMS were modified with allylamine plasma to form amine groups on the surface prior to the covalent grafting of succinimdyl ester-functionalized HPGs. The anionic functionality of the PDMS channel matrices was developed by altering the number of carboxyl groups present on the HPGs. The grafting of HPGs onto PDMS plates was investigated via contact angle measurement and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), while the grafting of the inside channel was investigated by electroosmotic flow (EOF) measurements. The charge density on grafted HPG was optimized to minimize the nonspecific protein adsorption and increase the selective capture of positively charged proteins. A proof-of-concept device was fabricated on PDMS and demonstrated that the device selectively captures positively charged protein (avidin) from a mixture of bovine serum albumin (BSA)-avidin at pH 7.4 in phosphate buffered saline (PBS). In order to increase the capture efficiency of the proteins in this PDMS-based device, pillar arrays have been fabricated within the channel. As a demonstration, the new device separated two proteins with an avidin capture efficiency of 100 ± 2.95% per 3 min from a 0.02 mg/ml protein solution (avidin:BSA wt ratio: 1:1). This new microfluidic-based device shows a great deal of promise as a tool for protein capture and analysis.     

Cell motion and recovery in a two-stream microfluidic device

The motion of cells in a two-stream microfluidic device designed to extract cryoprotective agents from cell suspensions was tested under a range of conditions. Jurkat cells (lymphoblasts) in a 10% dimethylsulfoxide solution were driven in parallel with phosphate-buffered saline solution wash streams through single rectangular channel sections and multiple sections in series. The influence of cell-stream flow rate and cell volume fraction (CVF) on cell viability and recovery were examined. The channel depth was 500 μm, and average cell stream velocity within the channels was varied from 3.6 to 8.5 mm/s corresponding with cell stream Reynolds numbers of 2.6–6.0. Cell viability measured at device outlets was high for all cases examined indicating no significant cell damage within the device. Downstream of a single stage, cell recoveries measured 90–100% for average cell stream velocities ≥6 mm/s and for CVFs up to 20%. Cell recovery downstream of multistage devices also measured 90–100% after a critical device population time. This time was found to be five times the average cell residence time within the device. The measured recovery values were significantly larger than those typically obtained using conventional cell washing methods.     

A polymeric micro-optical system for the spatial monitoring in two-phase microfluidics

This article presents a polymeric micro-optical system that consists of two coupled miniaturized devices for spatially distributed characterization of microfluidic two-phase phenomena exploiting multiwavelength optical signals. The input device implements four optical windows (slits) which are superimposed on the centerline of a microfluidic serpentine channel and illuminate specific locations of the microchannel. The flow-related information is then collected by an ad hoc polymeric micro-optical output device that guides and merges the spatially distributed information into a single output signal, which maintains memory of the spatial coordinates by using the wavelengths as fingerprints of the slits’ position in the microfluidic channel. Both micro-optical devices were designed, simulated, and characterized in static and dynamic conditions. Experiments on two-phase (air and ethanol) flow were carried out by applying constant and periodic flow rate functions. In both cases, the system was proved to be efficient in capturing the spatial–temporal dynamics of flow profiles.     

Novel surface acoustic wave (SAW)-driven closed PDMS flow chamber

In this article, we demonstrate a novel microfluidic flow chamber driven by surface acoustic waves. Our device is a closed loop channel with an integrated acoustic micropump without external fluidic connections that allows for the investigation of small fluid samples in a continuous flow. The fabrication of the channels is particularly simple and uses standard milling and PDMS molding. The micropump consists of gold electrodes deposited on a piezoelectric substrate employing photolithography. We show that the pump generates a pressure-driven Poiseuille flow, investigate the acoustic actuation mechanism, characterize the flow profile for different channel geometries, and evaluate the driving pressure, efficiency and response time of the acoustic micropump. The fast response time of our pump permits the generation of non-stationary flows. To demonstrate the versatility of the device, we have pumped a red blood cell suspension at a physiological rate of 60?beats/min.